Preserving cellular energy stores by inhibiting poly-ADP-ribosylation may represent a key neuroprotective strategy for the treatment of traumatic brain injury (TBI). However, poly-ADP-ribosylation may also serve beneficial roles after TBI by participating in the maintenance of genomic integrity, regulation of RNA transcription, and excitatory amino acid-mediated memory acquisition. These dichotomous roles likely depend upon the cellular compartment and specific poly-ADP-ribosylated protein targets, warranting rigorous examination in experimental models, prior to implementation of clinical treatment strategies targeting poly- ADP-ribosylation after acute brain injury. Our hypotheses are that: 1) acutely after brain injury, supra- physiologic poly-ADP-ribosylation is detrimental because it disturbs cellular energetics by depletion of NAD and ATP, leading to mitochondrial failure and cell death, and 2) chronically after brain injury, physiologic poly-ADP-ribosylation is beneficial because it facilitates DNA repair, regulates RNA transcription, and is important in processes related to memory. Our specific aims are to: 1) Further explore the organelle-specific effects of novel poly(ADP-ribose) polymerase (PARP) inhibitors after experimental TBI in vivo using specific PARP inhibitors, transgenic mice, and a targeted proteomics approach that identifies PARP substrates in nuclear, mitochondrial, and cytosolic compartments, 2) Further explore the neuroprotective and chronic effects of novel PARP inhibitors after TBI using a comprehensive battery of neuropathologic outcome tests evaluating structural and functional brain damage, and 3) Test the hypothesis that PARPactivation occurs in humans after TBI by applying a novel ELISA-based method for quantifying poly(ADP-ribose)-modified proteins in cerebrospinal fluid from humans with TBI, and identifying PARP substrates using a targeted proteomic approach, to establish a clinical basis for further development and a means for therapeutic drug monitoring of PARP inhibitors. TBI strikes without warning and is a major cause of morbidity and mortality in humans. Energy failure contributes to morbidity and mortality with only a few nonspecific therapies available. Specific aims will be integrated to completely define cellular proteins post-translationally modified by poly- ADP-ribosylation, to better define the role of PARPin normalcy and disease, and to aid in the development of novel cell compartment-specific agents targeting energy failure-induced cell death after TBI.